A framework for the systematic evaluation of a novel cigarette filter technology.

Any functional change in cigarette filter design warrants a rigorous assessment to ensure comparability to existing filter functionality. This study compares the functionality of a standard CA filter with a novel cellulose-based alternative using a combination of emissions, in silico approaches, pre-clinical assessments and behavioural studies. We assess the challenges faced with a significant filtration change, the substantiation of this change and the limitations of such assessments. We explore cigarette emission chemical profiles; assess the potential toxicological impacts (in vitro and statistical modelling) of the differing chemical profiles of cigarette smoke aerosol resulting from the respective filter types; and, finally investigate the behavioural aspects associated with use of the novel filter as compared to the traditional one. The aim of the study was to establish a weight of evidence assessment framework for the comprehensive evaluation of a novel cigarette filter design as part of robust stewardship approach. The data show comparability to a standard CA filter across all assessments and highlight potential areas of investigation for future novel filter product iterations. The approach demonstrates the applicability of a comprehensive step-wise assessment framework to identify any potential increased toxicant emissions and exposures associated with using the novel filter.

Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening.

BACKGROUND: The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI). METHODS: Whole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out. RESULTS: The results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected. CONCLUSIONS: Overall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks.

Genotoxicity evaluation of individual cigarette smoke toxicants using the in vitro γH2AX assay by high content screening.

Cigarette smoke is a complex mixture consisting of more than 5600 identified chemical constituents of which approximately 150 have been identified so far as "tobacco smoke toxicants". Proposals made by the World Health Organisation Framework Convention on Tobacco Control mandate the lowering of nine tobacco smoke priority toxicants, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), and benzo[a]pyrene (B[a]P) and monitoring the levels of a further nine including cadmium. Here, we evaluated the genotoxic potential in human bronchial epithelial BEAS-2B cells of four cigarette smoke toxicants; NNK, NNN, B[a]P and cadmium using the novel in vitro γH2AX assay by High Content Screening (HCS). We also examined the genotoxicity of binary mixtures of NNK and NNN reporting their relative contribution to the genotoxic end-point. The results of this preliminary assessment showed that the in vitro γH2AX assay by HCS could be used as a pre-screening tool to detect and quantify the genotoxicity effect of cigarette smoke toxicants individually and in binary mixture. Moreover, the data produced could contribute to the prioritisation of toxicant reduction research in modified tobacco products.

Assessment of the in vitro γH2AX assay by High Content Screening as a novel genotoxicity test.

The γH2AX assay is widely used as a marker of DNA damage in multiple scientific fields such as cancer biomarker, clinical studies and radiation biology. In particular, the in vitro γH2AX assay has been suggested as a novel in vitro genotoxicity test with potential as a pre-screening tool. However, to date, limited assessments have been carried out to evaluate the sensitivity, specificity and accuracy of the in vitro γH2AX assay. In this study, the microscopy-based system combining automated cellular image acquisition with software quantification for High Content Screening (HCS) has been used for the first time to evaluate the in vitro γH2AX assay. A panel of well-characterised genotoxic and non-genotoxic compounds was selected to assess the performance of the in vitro γH2AX assay in the human bronchial epithelial cell line BEAS-2B. The results obtained during this preliminary assessment indicate that the in vitro γH2AX assay has a high accuracy (86%) as a result of high sensitivity and specificity (86-92% and 80-88% respectively). Our data highlight the potential for γH2AX detection in HCS as a complement to the current regulatory genotoxicity battery of in vitro assays. We therefore recommend more comprehensive assessments to confirm the performance of the in vitro γH2AX assay by HCS with a more extensive set of compounds.

Metabolic characterization of cell systems used in in vitro toxicology testing: lung cell system BEAS-2B as a working example.

The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.

γH2AX as a novel endpoint to detect DNA damage: applications for the assessment of the in vitro genotoxicity of cigarette smoke.

Histone H2AX is rapidly phosphorylated to become γH2AX after exposure to DNA-damaging agents that cause double-strand DNA breaks (DSBs). γH2AX can be detected and quantified by numerous methods, giving a direct correlation with the number of DSBs. This relationship has made γH2AX an increasingly utilised endpoint in multiple scientific fields since its discovery in 1998. Applications include its use in pre-clinical drug assessment, as a biomarker of DNA damage and in in vitro mechanistic studies. Here, we review current in vitro regulatory and non-regulatory genotoxicity assays proposing the γH2AX assay as a potential complement to the current test battery. Additionally, we evaluate the use of the γH2AX assay to measure DSBs in vitro in tobacco product testing.